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. 2007 Aug 21;104(34):13666-71.
doi: 10.1073/pnas.0706192104. Epub 2007 Aug 14.

One at a time, live tracking of NGF axonal transport using quantum dots

Bianxiao Cui  1 , Chengbiao Wu, Liang Chen, Alfredo Ramirez, Elaine L Bearer, Wei-Ping Li, William C Mobley, Steven Chu
Affiliations

One at a time, live tracking of NGF axonal transport using quantum dots

Bianxiao Cui et al. Proc Natl Acad Sci U S A. .

Abstract

Retrograde axonal transport of nerve growth factor (NGF) signals is critical for the survival, differentiation, and maintenance of peripheral sympathetic and sensory neurons and basal forebrain cholinergic neurons. However, the mechanisms by which the NGF signal is propagated from the axon terminal to the cell body are yet to be fully elucidated. To gain insight into the mechanisms, we used quantum dot-labeled NGF (QD-NGF) to track the movement of NGF in real time in compartmentalized culture of rat dorsal root ganglion (DRG) neurons. Our studies showed that active transport of NGF within the axons was characterized by rapid, unidirectional movements interrupted by frequent pauses. Almost all movements were retrograde, but short-distance anterograde movements were occasionally observed. Surprisingly, quantitative analysis at the single molecule level demonstrated that the majority of NGF-containing endosomes contained only a single NGF dimer. Electron microscopic analysis of axonal vesicles carrying QD-NGF confirmed this finding. The majority of QD-NGF was found to localize in vesicles 50-150 nm in diameter with a single lumen and no visible intralumenal membranous components. Our findings point to the possibility that a single NGF dimer is sufficient to sustain signaling during retrograde axonal transport to the cell body.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Characterization of QD-NGF biological activity. (A) PC12 cells treated with 0.4 nM of QD-NGF complex for 3 h at 37°C showed bright punctuate fluorescence signal even after acid wash (Left). PC12 cells treated with the same concentration of QD and native NGF mixture showed fewer if any fluorescent puncta (Right). (B) Western blot analysis of phosphorylated TrkA (p-TrkA), phosphorylated Erk1/2 (p-Erk), and phosphorylated Akt (p-Akt) protein levels in PC12 cells in response to the treatment of native NGF, BtNGF, and QD-NGF. The phosphorylation of TrkA peaked at 5 min and decreased slightly at 30 min. Erk1/2 and Akt showed similar level of phosphorylation at 5 min and 30 min. The blots were also probed with antibodies sensitive to total Erk1/2 and total Akt to show equal loading of cell lysate proteins. Application of native NGF, BtNGF and QD-NGF at the same final concentration (2 nM) seemed to activate TrkA, Erk1/2, and Akt to a similar extent. (C) Illustration of QD-NGF bioactivity in PC12-cells. Two days treatment with 0.8 nM QD-NGF stimulated neurite outgrowth in PC12 cells. (D) Quantitative dose–response of PC12 cells to NGF, BtNGF and QD-NGF. The percentage of cells bearing neurites was counted after 2 days of continuous exposure to various concentrations of NGF, BtNGF and QD-NGF. (E) Schematic drawing of a QD-NGF bound to dimerized TrkA receptors (Left) and addition of QD-NGF to the DA compartment of the three-chamber DRG neuron culture (CAMP10, Right). DA, distal axon; PA, proximal axon; CB, cell body. (F) (Upper) Representative live fluorescence images of DRG neuron axons or cell bodies 2 h after the addition of 4 nM QD-NGF to the DA chamber. (Lower) Images are the results of these fluorescence images superimposed with their corresponding bright-field images. QD-NGF seems to bind all axons in distal axon chamber. However, only a small portion of the cell bodies and proximal axons are shown to have QD fluorescence, reflecting the fact that not all cell bodies extend their axons into the distal axon compartment.
Fig. 2.
Fig. 2.
Live imaging reveals that endosomes containing QD-NGF exhibit stop-and-go motion (see real-time SI Movies 1–5). (A) A typical axon showing four moving endosomes that contained QD-NGF in pseudocolor. The background fluorescence outlines the axon in this picture. (B) Trajectory of a typical endosome, showing a switch between moving and pausing. (C) Trajectories of 120 endosomes showing that the moving speed and the duration of pauses vary greatly from one endosome to another. (D) The average speed of endosomes containing QD-NGF varied from 0.2 to 3 μm/s. (E) Comparison of endosomes moving in three different axons. The trajectories of endosomes within the same axon are denoted with the same color (red, blue or green). The variability between different axons is significantly larger than the spread of velocities in the same group.
Fig. 3.
Fig. 3.
Transport dynamics and concentration dependence of QD-NGF containing endosomes. (A) Time-lapse video images (after background subtraction) of endosomes traveling on the same axon. Five endosomes were visible at the beginning of the video recording, and the sixth endosome came into the field of view after 6 s. The endosomes are numbered according to their axonal positions at the start of the video recording. After 4 s, two fast moving endosomes (no. 2 and no. 5) passed the slower ones (no. 1 and no. 4). The white arrow indicates that direction of motion was toward the cell body. (B) Trajectories of fifteen endosomes moving in the same axon through the same field of view. The majority of endosomes move independently (black circles). Endosomes moving together or passing another endosomes are shown in red and green for clarity. The blue arrows indicate the places where some trajectories paused at the same axonal location. (C) The number of endosomes in a fixed length of axon increases with QD-NGF concentration. Typical images showing the density of endosomes increases with QD-NGF concentration (see real-time SI Movies 1–5). At 20 nM concentration, the fluorescence intensity of individual endosomes contained QD-NGF increased significantly. (D) Average number of endosomes per 1 mm of axons increases with increased QD-NGF concentration ranging from 0.2 to 20 nM.
Fig. 4.
Fig. 4.
Quantification of the number of QD-NGFs contained in individual endosomes. (A) “Photo-blinking” of a single QD particle. A time sequenced (every 0.1 s) images of a QD-NGF containing moving endosome. The sudden disappearance of QD fluorescence for 0.7 s in the middle is due to the intrinsic photo-blinking property of QDs. Time trace of the fluorescence intensity of the endosome is shown in B. The fluorescence intensity of the endosome is consistent with the intensity of a single QD particle. The sudden decrease of the fluorescence signal to background levels (“blinking”) is the signature of a single QD particle. (C) Distribution of the number of QD particles contained in endosomes when QD was mixed with BtNGF at molar ratios of 1.2:1 and 10:1. The number of QD particles in each endosome was quantified by measuring both the blinking events and the fluorescence intensity. (D) Distribution of the number of QD particles in endosome at the QD-NGF concentration of 0.2, 2 and 20 nM. The majority of endosomes contained a single QD-NGF at 0.2 and 2 nM. At 20 nM, ≈30% of endosomes contained just one QD-NGF and the rest contained two or more QD-NGF conjugates.
Fig. 5.
Fig. 5.
EM and immunostaining analysis of endosomes containing QD-NGF. DRG neurons treated with QD-NGF were fixed, embedded and processed for ultrathin sectioning. (A) A representative EM image of a cross-section of the proximal axon portion showing that it contained many small vesicles (50–150 nm in diameter). One vesicle contained QD (arrows). (B) Out of 84 QD containing vesicles, most had 1–2 QD particles. (C) A representative image of DRG neurons cultured inside a microfluidic device. Axons were able to extend across two columns of microgrooves into the distal axon chamber. (D) After QD-NGF was added to the DA chamber for 2 h, all chambers were rinsed and fixed for immunostaining by using the indicated primary antibodies. A secondary antibody-Alexa 488 conjugate (green) was used to reveal these primary antibodies (green). We confined our observations to the sections of axons leading to the cell body in the microgrooves. The colocalization (yellow) between QD-NGF (red) and Trk, Rab5B and pErk1/2 (all green) is extensive (three panels in Upper). When QD was omitted, we were unable to detect a red signal but the green signals for the other markers such as Trk (Lower) remained.
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